Although mouse spermatozoa were "successfully" frozen more than a decade ago, the fundamental cryobiological factors that affect the ability of spermatozoa to survive with full function after freezing-thawing remain largely unknown. Current methods used to cryopreserve mouse spermatozoa have markedly improved for hybrid strains, but do not routinely work for inbred strains. Germplasm repositories are being established as a method to preserve valuable genetically engineered mouse (GEM) strains. However, the inability to cryopreserve sperm from inbred strains poses a significant problem since the majority of GEM is maintained on inbred lines. Freezing and storing sperm could most efficiently preserve the majority of these newly created strains where the strain or mutation of interest can be adequately preserved by haploid germplasm. However, in spite of the fact that several methods have been reported to produce moderate success in mouse sperm cryopreservation, they are still inadequate for safe preservation of all mouse strains. The thesis of this proposal is that an understanding of the fundamental cryobiology of mouse sperm from a number of commonly used inbred strains fro the production of GEM will permit the development of optimal procedures for cryopreservation. Our studies to date with mouse sperm have provided basic information regarding the permeability and the osmotic and mechanical properties of these cells and have resulted in significant improvements in hybrid mouse strain sperm cryopreservation. Here we propose to expand this work to inbred strains. The experimental approach proposed is to: (a) perform experiments necessary to determine the effects of the cryopreservation process on acrosomal integrity of mouse sperm and to develop methods to prevent premature acrosomal loss, (b) determine the membrane permeability characteristics of mouse sperm from several genetic backgrounds to water and various cryoprotectant solutes, to predict optimum conditions for cooling and warming, and (c) determine optimal cryoprotectant media composition for spermatozoa from various mouse genetic backgrounds. Information gained from these experiments will develop improved procedures of mouse germplasm banking, provide important information regarding the genetic variability in mouse sperm cryopreservation and develop a foundation for improvements in rederivation efficiency when using frozen semen.